Hepatitis C Virus Replication and Intracellular Membrane Rearrangement

丙型肝炎病毒复制和细胞内膜重排

• 批准号: 7938201
• 负责人: Kouacou V. KONAN
• 金额: $ 5.4万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-21 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938201
• 关键词: Address; Biological Assay; Cells; Complex; DNA Sequence Rearrangement; Data; Detection; Early Endosome; Electron Microscopy; Endocytosis; Endoplasmic Reticulum; Genome; Hepatitis C; Hepatitis C virus; Immunoprecipitation; In Vitro; Integration Host Factors; Internet; Intracellular Membranes; Laboratories; Life Cycle Stages; Malignant neoplasm of liver; Mass Spectrum Analysis; Membrane; Mutation; Nonstructural Protein; Play; Proteins; Reporting; Role; Site; Staining method; Stains; Testing; Viral; Viral Genome; Viral Proteins; Virus Replication; base; novel; protein profiling; rab5A Protein; scaffold

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a leading cause of liver cancer worldwide. During infection, HCV induces the formation of rearranged membrane structures termed web. These membranes are also observed in vitro following expression of HCV NS4B protein alone. Various reports have shown that the web contains HCV nonstructural proteins as well as the viral genome, suggesting that this novel membrane might be the site where HCV genome replication occurs. This proposal will test the following hypotheses: (i) the web plays a critical role in HCV genome replication, (ii) the web contains several host factors, including Rab5, which play a direct or indirect role in HCV life cycle. To test these hypotheses, we propose the following Specific Aims: Aim I. Test whether the web plays a critical role in HCV genome replication. It is widely believed that the web provides the scaffold for HCV genome replication. However, there is no direct experimental evidence to prove this hypothesis. Our current assay for web formation consists of co-localization of punctate NS4B staining with endogenous Rab5B protein. This assay is generally confirmed by electron microscopy detection of the web. In this Aim, we will (IA) introduce mutation(s) in NS4B to abolish web formation and (IB) determine whether abolishing the web inhibits HCV genome replication. We will (1C) test whether NS4B oligomerization plays a role in web formation and genome replication. Aim II. Determine the significance of the presence of Rab5 protein in the web. Previous reports have shown that the web-inducing protein, NS4B, is associated mostly with the endoplasmic reticulum (ER) compartment. Our laboratory has recently found that early endosome markers, Rab5 and EEA1, are associated with web-inducing NS4B. In addition, Rab5 was found to interact with NS4B. In this Aim, we will (IIA) examine how Rab5 gets incorporated into the web. We will (IIB) identify the Rab5-associated proteins interacting with HCV NS4B. Finally, we will (IIC) determine whether the web alters endocytosis in the cell. Aim III. Determine the protein profile of the web. The association of web-inducing NS4B protein with ER and EE marker, Rab5, suggests that this novel membrane has a complex origin and composition. In our preliminary results, we have used lodixanol (Optiprep) gradient to isolate membrane fractions enriched in viral proteins involved in web formation and genome replication. In this Aim, we will (IIIA) combine Optiprep gradient with immunoprecipitation to isolate web-enriched membranes. We will (IIIB) use mass spectrometry to examine the protein profile of the immuno-isolated membrane. We hope that these studies will address the role of HCV-associated ultrastructural changes in the viral genome replication and viral persistence.

描述（由申请人提供）：丙型肝炎病毒（HCV）是全世界肝癌的主要原因。在感染过程中，HCV 诱导形成称为网的重新排列的膜结构。在单独表达 HCV NS4B 蛋白后，也在体外观察到这些膜。各种报告表明，该网络包含 HCV 非结构蛋白以及病毒基因组，表明这种新型膜可能是 HCV 基因组复制发生的位点。该提案将测试以下假设：（i）网络在HCV基因组复制中发挥关键作用，（ii）网络包含多个宿主因子，包括Rab5，它们在HCV生命周期中发挥直接或间接作用。为了检验这些假设，我们提出以下具体目标： 目标 I. 测试网络是否在 HCV 基因组复制中发挥关键作用。人们普遍认为网络为 HCV 基因组复制提供了支架。然而，没有直接的实验证据证明这一假设。我们目前的网络形成分析包括点状 NS4B 染色与内源 Rab5B 蛋白的共定位。该测定通常通过网的电子显微镜检测来证实。在此目标中，我们将 (IA) 在 NS4B 中引入突变以消除网的形成，并 (IB) 确定消除网是否会抑制 HCV 基因组复制。我们将 (1C) 测试 NS4B 寡聚化是否在网络形成和基因组复制中发挥作用。 目标二。 确定 Rab5 蛋白在网络中存在的重要性。之前的报告表明，网诱导蛋白 NS4B 主要与内质网 (ER) 区室相关。我们的实验室最近发现早期内体标记 Rab5 和 EEA1 与 web 诱导的 NS4B 相关。此外，还发现 Rab5 与 NS4B 相互作用。在此目标中，我们将 (IIA) 研究 Rab5 如何融入网络。我们将 (IIB) 鉴定与 HCV NS4B 相互作用的 Rab5 相关蛋白。最后，我们将（IIC）确定网络是否改变细胞中的内吞作用。 目标三。 确定网络的蛋白质谱。网诱导 NS4B 蛋白与 ER 和 EE 标记 Rab5 的关联表明这种新型膜具有复杂的起源和组成。在我们的初步结果中，我们使用洛迪克沙醇 (Optiprep) 梯度来分离富含参与网络形成和基因组复制的病毒蛋白的膜组分。在此目标中，我们将 (IIIA) 将 Optiprep 梯度与免疫沉淀相结合来分离网络富集的膜。我们将 (IIIB) 使用质谱法检查免疫隔离膜的蛋白质谱。 我们希望这些研究能够解决 HCV 相关超微结构变化在病毒基因组复制和病毒持久性中的作用。

项目成果

Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

• DOI: 10.1016/j.virol.2009.07.033
• 发表时间: 2009-10-10
• 期刊: VIROLOGY
• 影响因子: 3.7
• 作者: Aligo, Jason;Jia, Shuaizheng;Manna, David;Konan, Kouacou V.
• 通讯作者: Konan, Kouacou V.

Endocytic Rab proteins are required for hepatitis C virus replication complex formation.

• DOI: 10.1016/j.virol.2009.11.034
• 发表时间: 2010-03-01
• 期刊: VIROLOGY
• 影响因子: 3.7
• 作者: Manna, David;Aligo, Jason;Xu, Chenjia;Park, Wei Sun;Koc, Hasan;Heo, Won Do;Konan, Kouacou V.
• 通讯作者: Konan, Kouacou V.